Full-length HIV-1 RNA needs to be exported from the nucleus to carry out its functions, including serving as a template for Gag/Gag-Pol translation and as a genome in the newly assembled virion. To study HIV-1 RNA packaging, we have developed a novel assay to directly visualize viral RNA in the particles at single-RNA-molecule sensitivity. This assay reveals that most HIV-1 particles contain viral RNA. Although it is known that retroviral RNAs packaged into particles are dimeric, standard biochemical assays cannot determine the number of RNA molecules in one particle; our results provide evidence to support the long-standing assumption that two RNA molecules (one dimer) are packaged into a particle. To better understand the mechanisms of RNA packaging, we have performed genetic, biochemical, and imaging analyses to show that HIV-1 RNA uses base-pairing of the DIS sequences to select its copackaged RNA partner, and this process occurs in the cytoplasm of the producer cell prior to RNA packaging into particles. We and others have observed that HIV-1 RNA can be manipulated to deviate from using the CRM1-mediated nuclear export pathway and, instead, be exported via the NXF1 pathway, which is used by most cellular mRNAs. However, we found that RNA molecules exported through these two pathways do not copackage efficiently, indicating that the export pathway affects subcellular localization of these RNAs. Our current efforts in this project are focused on examining the dynamics of HIV-1 RNA export using high-speed, high-resolution microscopy to detect export of a single HIV-1 RNA through the nuclear pore complex. We are investigating the kinetics of export via the CRM1 or NXF1 pathways and exploring whether different export pathways use different nuclear pore complexes. We are also investigating the mechanisms that HIV-1 uses to regulate which and how many RNAs are packaged. Additionally, we are using an RNA-binding protein to redirect HIV-1 RNA-packaging specificity, and we are using super-resolution microscopy to study the RNA structure in viral particles. These studies will allow us to understand several questions that are fundamentally important to HIV replication, which can be used to generate new strategies to block the spread of HIV. [Corresponds to Hu Project 2 in the October 2011 site visit report of the HIV Drug Resistance Program]